Streptococcus pneumoniae are Gram-positive, lancet shaped cocci that are usually seen in pairs (diplococci), but also in short chains or as single cells. They grow readily on blood agar plates with glistening colonies and display alpha hemolysis unless grown anaerobically where they show beta hemolysis. They are sensitive to bile salts that can break down the cell wall with the presence of the cells' own enzyme, autolysin. The organism is an aerotolerant anaerobe and is fastidious in that it has complex nutritional requirements.
The cells of most pneumococcal serotypes have a capsule which is a polysaccharide coating surrounding each cell. This capsule is a determinant of virulence in humans because it interferes with phagocytosis by preventing antibodies from attaching to the bacterial cells. There are currently 90 capsular serotypes identified, with 23 serotypes responsible for about 90% of invasive disease. As a vaccine the polysaccharide can confer a reasonable degree of immunity to S. pneumoniae in individuals with developed or unimpaired immune systems. However, when the polysaccharide is conjugated with a high molecular weight protein such as CRM197 and formulated into a vaccine containing conjugates of multiple serotypes, such conjugate vaccines allow for an immune response in infants and elderly who are also most at risk for pneumococcal infections.
The capsular polysaccharide for each S. pneumoniae serotype utilized for vaccine products is produced by growing the organism in liquid medium. The population of the organism is often scaled up from a seed vial to seed bottles and passed through one or more seed fermentors of increasing volume until production scale fermentation volumes are reached. The end of the growth cycle can be determined by one of several means, at which point the cells are lysed through the addition of a detergent or other reagent which aids in the cell wall breakdown and release of autolysin which causes cellular lysis when the cells reach stationary phase. The broth is then harvested for downstream (purification) processing. The major contaminants are cellular proteins, nucleic acids, C-polysaccharide and medium components.
For most of the serotypes for the currently marketed 7-valent pneumococcal conjugate (7vPnC) vaccine (PREVNAR®), as well as the newly developed 13-valent pneumococcal conjugate (13vPnC) vaccine, the current purification process requires sixteen steps involving many expensive, labor intensive and technologically demanding operations, such as chromatography and multiple membrane separations. Previous attempts at improving purification processes for S. pneumoniae polysaccharides have included, for example, pH manipulation during fermentation and recovery (see U.S. Patent App. Pub. No. 2006/0228381) and solvent and detergent precipitation. However, the removal of impurities in these processes is still spread over many labor intensive and costly steps. Protein level is the most problematic specification to meet due to the physical and chemical properties of the soluble proteins.
Thus, there is a need for a simplified purification process to reduce the soluble protein levels in S. pneumoniae lysates and eliminate inefficiencies of the current purification process to produce substantially purified capsular polysaccharides suitable for incorporation into pneumococcal conjugate vaccines.